Fibroblast growth factor (FGF) signaling plays pleiotropic roles in mammalian development and metabolism, and disease. The paracrine FGF1, FGF4, FGF7, FGF8, and FGF9 subfamilies play essential roles in spermatogenesis, mesoderm induction, somitogenesis, organogenesis, and pattern formation, whereas the FGF19 subfamily acts in an endocrine fashion to regulate major metabolic processes including glucose, lipid, cholesterol, and bile acid metabolism, and phosphate/vitamin D homeostasis. The diverse activities of FGFs are transmitted by the FGF receptor (FGFR) subfamily of receptor tyrosine kinases (RTKs). Perturbed FGF signaling leads to numerous human diseases, including skeletal, reproductive syndromes, hearing loss, renal phosphate wasting, neurodegenerative disorders, and cancer. Several paracrine FGFs and all the endocrine FGFs are being pursued for drug development. The four specific aims of this competing renewal are: I. Characterize the structural basis by which epithelially-expressed FGF4 and FGF9 subfamilies attain their specificity towards mesenchymally-expressed FGFRc isoforms. II. Elucidate the structural basis by which a/bKlotho co-receptors promote signaling by the endocrine FGFs. III. Dissect the role of A-loop tyrosine phosphorylation in the hyperactivation of FGFR tyrosine kinase by pathogenic gain-of-function mutations. IV. Elucidate the structural basis by which FGFR recruits and phosphorylates FRS2a. Recombinant protein expression and engineering, x-ray crystallography, Surface Plasmon Resonance (SPR) spectroscopy, isothermal titration calorimetry (ITC), steady-state kinetics analysis, and time-resolved mass spectrometry will be used to accomplish the Specific Aims of this proposal. The structural and biophysical/biochemical results obtained will also be validated using cell- and animal-based assays. The data obtained under Aim I should provide molecular insights into the roles of paracrine FGFs in embryonic development and also facilitate the discovery of drugs for tissue repair and bioengineering, promotion of self- renewal and differentiation of human embryonic stem cells for cell-replacement therapy. The data generated under Aim II should enhance our understanding of the role of endocrine FGFs in human metabolism and provide blueprints for drug discovery for major human diseases including diabetes, obesity, hypercholesterolemia, colon cancer, and chronic kidney disease, most of which represent a huge burden on public health. The results of Aim III will enhance our understanding of the mechanism of action of pathogenic mutations in FGFRs and other RTKs as well as the regulation of tyrosine kinase activity of the entire RTK superfamily. Since substrate recruitment and phosphorylation by RTKs is a general event in RTK signaling, the mechanistic insights gained under Aim IV will also be directly applicable to the entire RTK superfamily.